Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

Glucocorticoids are known to increase the cyclic AMP response to parathyroid hormone (PTH) in cultured bone organs or bone cells. Using the osteoblast-like cell line ROS 17/2.8, which possesses receptors for both PTH and glucocorticoids, we investigated which component of the complex hormone receptor-guanine nucleotide regulatory unit--adenylate cyclase was affected by dexamethasone treatment. In response to PTH, isoproterenol or forskolin, a compound that is supposed to act directly on the catalytic unit, cyclic AMP production by intact cells and adenylate cyclase activity in purified plasma membrane were markedly increased by dexamethasone. Whereas NaF, guanosine 5'-[beta gamma-imido]triphosphate and Mn/ stimulated adenylate cyclase activity were similarly enhanced in membranes isolated from glucocorticoid-treated cells, the activity of the stimulatory guanine nucleotide regulatory unit, as assessed by reconstitution into membranes from the CYC- clone, which is genetically devoid of this component, was not altered. Thus in osteoblast-like cells dexamethasone appears to increase cyclic AMP synthesis by influencing the catalytic unit. Moreover, since it has been reported that glucocorticoids may produce changes in cell calcium metabolism, we evaluated cytoplasmic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ stores mobilizable by the bivalent-cationophore ionomycin, by using the intracellular fluorescent indicator Quin-2. The results indicated that dexamethasone treatment did not influence [Ca2+]i but markedly decreased ionomycin-releasable Ca2+ stores.     

Activation of lipolysis and cyclic AMP accumulation in rabbit adipocytes by isoproterenol in the presence of forskolin or pertussis toxin

Adipocytes from rabbits are relatively insensitive to catecholamines or forskolin. However, the combination of catecholamines plus forskolin increased cyclic AMP accumulation and lipolysis much more than either agent alone. Pertussis toxin treatment also restored sensitivity to catecholamines. No defect in activation by catecholamines of adenylate cyclase was seen in isolated membranes incubated in the presence of GTP. Rabbit adipocytes appear to have an excess of the inhibitory guanine nucleotide binding protein (Ni). However, in plasma membranes this protein appeared to be relatively inactive as there was an activation of adenylate cyclase activity by catecholamines in the presence of GTP. These data suggest that in intact rabbit adipocytes catecholamines and forskolin are ineffective as stimulators of adenylate cyclase due to an excess of inhibitory guanine nucleotide binding proteins.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Translocation of phospholipid/Ca2+-dependent protein kinase in B-lymphocytes activated by phorbol ester or cross-linking of membrane immunoglobulin.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

Evidence that muscarinic receptors in islet cells are not coupled functionally to adenylate cyclase through the inhibitory guanine nucleotide binding protein (Ni)

The effect of muscarinic agonist on adenylate cyclase was investigated in neonatal islet cells and in a clonal pituitary cell line (GH4C1) following labelling of the intracellular ATP pool with [2,8 3H]adenine. In islet cells carbamylcholine was without effect on basal or glucagon-stimulated adenylate cyclase activity, measured as 3H cyclic AMP production, but inhibited 3H cyclic AMP production in the clonal pituitary cells. The involvement of the inhibitory guanine nucleotide binding protein of adenylate cyclase (Ni) was investigated by the use of the Bordetella pertussis exotoxin, islet activating protein (IAP). Pre-treatment of islet cells with IAP was without effect on adenylate cyclase following carbamylcholine but in the clonal pituitary line abolished the inhibition of 3H cyclic AMP production. It is concluded that in the islet cell, in contrast to the clonal pituitary cell, muscarinic receptors are not effectively coupled through Ni to inhibit adenylate cyclase.     

"Spare" beta-adrenergic receptors of rat white adipocyte membranes

The apparent equilibrium dissociation constants for the interaction of isoproterenol with beta-receptors and adenylate cyclase were determined under the same conditions in rat adipocyte membranes and were compared with the apparent dissociation constant for the interaction of isoproterenol with cyclic AMP accumulation in the adipocyte. From these determinations, it was calculated that the occupancy of less than 4% of the receptor population is required for half-maximal stimulation of adenylate cyclase in membranes and cyclic AMP accumulation in intact cells, provided that receptor-binding and adenylate cyclase assays are performed in the presence of guanine nucleotides. Since guanine nucleotides are also required for adenylate cyclase activation in intact cells, it is concluded that the beta-receptors of rat adipocytes are "spare" receptors.     

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.     

Desensitization of the epidermal adenylate cyclase system: agonists and phorbol esters desensitize by independent mechanisms.

Exposure of pig epidermis to adenylate cyclase stimulators results in receptor-specific desensitization. We investigated the nature of the agonist-induced desensitization, which was compared with the phorbol ester-induced, receptor-nonspecific desensitization. Both phorbol ester-induced desensitization and the agonist-induced desensitization were accompanied by an increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. The magnitude of the increase in the agonist-induced desensitization was parallel to the degree of the initial cyclic AMP accumulation; histamine and adenosine, which increase more cyclic AMP than epinephrine, resulted in a more marked increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. Similarly, epidermis desensitized to multiple receptors revealed more marked forskolin- and cholera toxin-induced cyclic AMP accumulations than epidermis desensitized to a single receptor. In contrast to the phorbol ester-induced desensitization, agonist-induced desensitization was not affected by the protein kinase C inhibitors H-7 and staurosporin. Further, agonist-induced desensitization was still inducible in phorbol ester-desensitized epidermis and vice versa. In contrast to the agonist-induced desensitization, which is accompanied by the preceding adenylate cyclase stimulation, no evidence for the stimulation of the adenylate cyclase during phorbol ester treatment was obtained. Neither agonist-induced desensitization nor phorbol ester-induced desensitization affected the content of inhibitory guanine nucleotide binding protein of the epidermis, which was monitored by the pertussis toxin (IAP)-catalyzed ADP ribosylation reaction. Our results indicate that agonist-induced desensitization and the phorbol ester-induced desensitization are independent of each other. Although both processes are characterized by increased forskolin- and toxin-induced cyclic AMP accumulations, the former is accompanied by initial cyclic AMP accumulation; the latter is not.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Age related decline in aluminum-activated human platelet adenylate cyclase: post-receptor changes in cyclic AMP second messenger signal amplification in normal aging and dementia of the Alzheimer type

Low, micromolar concentrations of aluminum (in the presence of NaF) were shown to strongly activate human platelet adenylate cyclase and provided a useful probe for evaluating cyclic AMP second messenger function distal to the receptor: The effect of normal aging and disease state on second messenger activity in man was studied by measurements of the aluminum-activated enzyme. A significant decline in aluminum-stimulated platelet adenylate cyclase activity in older, healthy subjects was observed. An age-associated decline in NaF-stimulated cyclic AMP synthesis was also demonstrated for normal, non-demented subjects. These findings suggest an age-associated lesion at the level of the guanine nucleotide regulatory protein/catalytic subunit of the adenylate cyclase complex. However, for patients with Alzheimer's disease no such decline in platelet adenylate cyclase activity was detected, and increased sensitivity to both aluminum and NaF was demonstrated.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.     

Somatostatin acts through G-proteins on dopaminergic adenylate cyclase in the caudate-putamen of the rat

Somatostatin was incubated in an adenylate cyclase assay of a particulate fraction of caudateputamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Somatostatin was able to enhance cyclic AMP formation in the presence of guanylylimidodiphosphate and guanosine-triphosphate. In contrast to this, somatostatin inhibited both dopamine and forskolin-stimulated cyclic AMP accumulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation. Somatostatin was found to antagonize these inhibitory effects of pertussis toxin and cholera toxin. The results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect dopaminergic adenylate cyclase activity.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta.

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.